Collecting‐duct‐principal‐cell‐select knockout (KO) of the mechanistic‐target‐of‐rapamycin (mTOR) alters sodium and acid/base homeostasis

Abstract

The mechanistic target of rapamycin (mTOR), a serine‐threonine kinase downstream of insulin receptor signaling and existing in a complex (mTORC2) with ancillary proteins, has been demonstrated to activate the renal epithelial sodium channel (ENaC). To further elucidate mTOR's role in the kidney collecting duct (CD), we generated CD‐principal‐cell‐select mTOR KO mice by Cre‐lox recombination using aquaporin‐2 (AQP2) promoter to drive Crerecombinase. A single injection of benzamil (ENaC antagonist) to male mice (n = 6/genotype) showed significantly reduced natriuretic response in the KO (32%) supporting lower ENaC activity. Urine was collected in metabolic cages while mice (n = 13/genotype) were fed a normal chow (1% NaCl) or a very low sodium (Na+) diet (<0.05%, LSD) for one week. Urine sodium concentrations were significantly higher on day 1 of the LSD in the KO (16.3 ± 1.8 versus 27.4 ± 4.3 mM, p = 0.024); but not on remaining days. Urine aldosterone (ng/d) rose 3–4 fold by day 7 with LSD in both genotypes, but was ~15–25% lower in the KO on most days. Mice were then divided to receive control diet or the LSD for an additional week and euthanized. Western blotting of cortex homogenates revealed a 20–40% reduction in the protein abundances of α, β‐and γ‐ENaC and aquaporin‐2 in the KO, regardless of diet. Furthermore, Rhbg (renal ammonia transporter) was ~65% lower in the KO. Blood chemistry showed significantly lower Na+ and higher bicarbonate (HCO3) concentrations in the KO under normal diet, and these differences were eliminated by the LSD. To test responses when aldosterone levels were clamped high, in Study 2, male mice were infused with aldosterone (20 mg/30 g·bw/d) by osmotic minipump for 3 days, then ½ of each genotype were switched to a high NaCl diet (4%) for 4 days. Clamping aldosterone high resulted in some “reversals” of phenotype, i.e., KO mice had significantly higher blood Na+ and lower HCO3 concentrations (relative to WT). This was associated with a modest relative reduction in K+ excretion and urine volume in the KO in the “aldosterone escape” phase, leading to an elevation in plasma K+ at the completion of the study. This may suggest relative aldosterone insensitivity in the KO. In sum, these studies support a role for CD‐principal cell mTOR in electrolyte and acid/base homeostasis. They also support a central role for mTOR in integrating growth factor and aldosterone actions at this site.Support or Funding InformationGeorgetown Medical Center Pilot Award (CE)Post‐Doctoral TL‐2 Award (MF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.